identity of environmental dna sequences using descriptions of four

ORIGINAL PAPER

Identity of environmental DNA sequences using descriptionsof four novel marine gregarine parasites, Polyplicariumn. gen. (Apicomplexa), from capitellid polychaetes

Kevin C. Wakeman & Brian S. Leander

Received: 21 September 2012 /Revised: 23 November 2012 /Accepted: 29 November 2012 /Published online: 18 April 2013# Senckenberg Gesellschaft für Naturforschung and Springer-Verlag Berlin Heidelberg 2013

Abstract Environmental PCR surveys of small-subunit(SSU) rDNA sequences are powerful approximations forthe overall diversity of microbial eukaryotes (protists) livingin specific marine habitats. However, many environmentalDNA sequences generated from these approaches have un-known cellular origins because they are not closely relatedto other sequences that were generated directly from fullycharacterized, identified organisms. The unidentifiedsequences from marine environments tend to belong topoorly understood groups of apicomplexan parasites, espe-cially gregarines. Single-cell PCR (SC-PCR) approaches onnewly discovered gregarines provide the evidence necessaryfor determining the cellular identities of SSU rDNA se-quence clades. In this study, the trophozoites of four novelgregarine morphotypes were isolated from the intestines oftwo different species of capitellid polychaetes collectedfrom the eastern Pacific Ocean (British Columbia, Canada).The trophozoites of each morphotype were characterizedusing light microscopy, scanning electron microscopy, andSSU rDNA sequences amplified from four, single-cell iso-lates from each of the four novel morphotypes described inthis study (16 new SSU rDNA sequences in total).Molecular phylogenetic analyses demonstrated five robustsubclades within a more inclusive clade that contained all 16new sequences and 5 environmental SSU rDNA sequences.The combination of SC-PCR approaches, molecular

phylogenetic analyses, and comparative morphology (1)illustrate the utility of SC-PCR approaches for distinguish-ing between different gregarine species, (2) demonstratethe cellular identity of a previously unidentified environ-mental SSU rDNA sequence clade, and (3) enable us toestablish four new species within one novel genus:Polyplicarium lacrimae n. gen. et sp. (type species), P.curvarae n. gen. et sp., P. translucidae n. gen. et sp., andP. citrusae n. gen. et sp.

Keywords Alveolata . Apicomplexa . Capitellidpolychaetes . Environmental DNA sequences . Marinegregarines . Molecular phylogeny . Polyplicarium

Introduction

Marine gregarine apicomplexans are a diverse but poorlyunderstood assemblage of endoparasites that infect the intes-tines and other extracellular spaces in a wide range ofmarine invertebrates (Grassé 1953; Levine 1971, 1976;Perkins et al. 2002). Only a tiny fraction of the knowndiversity of marine gregarines is represented in molecularphylogenetic datasets; the most widely explored marker sofar has been small-subunit (SSU) rDNA sequences (Leander2007; Leander and Keeling 2004; Leander et al. 2003, 2006;Rueckert et al. 2010; Rueckert and Leander 2008, 2009,2010; Rueckert et al. 2011a, b; Wakeman and Leander2012). Nonetheless, phylogenetic analyses of DNA sequen-ces used in tandem with high-resolution microscopy oftrophozoite stages has helped shape our understanding ofgregarine diversity and evolutionary history (Leander 2008).This approach has also been vital for the delimitation andidentification of different gregarine species and for estab-lishing the cellular identities of ambiguous environmental

K. C. Wakeman (*) :B. S. LeanderCanadian Institute for Advanced Research,Program in Integrated Microbial Biodiversity,Department of Zoology,University of British Columbia,#3529 6270 University Boulevard,Vancouver, BC, Canada V6T 1Z4e-mail: [email protected]

Mar Biodiv (2013) 43:133–147DOI 10.1007/s12526-012-0140-5

DNA sequences generated from several different PCR sur-veys of marine biodiversity (Berney et al. 2004; Cavalier-Smith 2004; Dawson and Pace 2002; Edgcomb et al. 2002;Leander and Ramey 2006; López-García et al. 2007;Moreira and López-García 2003; Stoeck and Epstein 2003;Stoeck et al. 2007; Takishita et al. 2005, 2007a, b).

Environmental PCR surveys targeting SSU rDNAsequences are informative approximations for the overallcomposition of species in an ecosystem, especially whenconsidering the vast assortment of uncultivated lineages ofmicrobial eukaryotes present in these systems (e.g., intertid-al areas, salt marshes, and deep sea hydrothermal vents).However, the cellular identities of numerous environmentalsequences generated from marine environments remain am-biguous. This is mainly due to an inability to establish thesister lineages of highly divergent sequences using molecu-lar phylogenetic datasets with only a limited sample of taxathat have also been characterized at the morphological level(Leander and Ramey 2006; Rueckert et al. 2011a, b). Thissituation has led some authors to conclude that the variationobserved in some SSU rDNA sequences represents novellineages of eukaryotic diversity that are largely or com-pletely unknown (Dawson and Pace 2002; López-Garcíaet al. 2007; Stoeck and Epstein 2003; Stoeck et al.2007). Other authors interpret ambiguous environmentalsequences as representing known species or more inclu-sive taxonomic groups that have yet to be characterizedat the molecular level (Cavalier-Smith 2004; Rueckert etal. 2011a, b). These contrasting interpretations are diffi-cult to evaluate when the molecular phylogenetic rela-tionships between lineages of interest are unresolved(Leander 2008).

The exploration of gregarine diversity using molecularphylogenetic data has established the cellular identities ofseveral different environmental sequence clades within theApicomplexa (Leander and Ramey 2006; Rueckert et al.2011a, b). The lifecycle of marine gregarine apicomplexansincludes a cyst stage that, in marine environments, is dis-persed in the sediment and eventually ingested by a newindividual host (Leander 2008; Vivier and Desportes 1990).The amplification of DNA sequences in environmental PCRsurveys suggest that the cysts of marine gregarines areprevalent in marine sediments and show that the extremedivergence of some gregarine SSU rDNA sequences makethem difficult to analyze (Cavalier-Smith 2004; Leander2007; Leander and Ramey 2006; Takishita et al. 2005,2007a, b). A study of gregarines isolated from the intestinesof crustaceans, for instance, demonstrated that the highlydivergent SSU rDNA sequences from these particular spe-cies were only identifiable in PCR surveys after establishingdirect links between the DNA sequences and other cellulartraits (e.g., trophozoite morphology). Therefore, species dis-covery surveys that aim to characterize novel organisms

using culture-independent methods to acquire data at bothmorphological and molecular levels provide the necessarycontext for identifying clades of ambiguous environmentalsequences.

Like most groups of microbial eukaryotes (protists),gregarine parasites are particularly prone to having a lowdegree of morphological diversity between different spe-cies and a high degree of morphological plasticity with-in species (Rueckert et al. 2011b); moreover, like otherparasites, gregarines have different life cycle stages thatvary considerably at the morphological level (e.g.,gametocysts, oocysts, and the developmental stages be-tween sporozoites and trophozoites). Therefore, usingextensive and tedious morphometric measurements todelimit one gregarine species from another is not onlyinadvisably time consuming for delimiting one speciesfrom another but also largely impenetrable, impractical,and misleading.

In this study, we discover and characterize the morphol-ogy and molecular phylogenetic markers of four novel spe-cies of Pacific marine gregarines isolated from the intestinesof two capitellid polychaetes, Notomastus tenuis andHeteromastus filiformis. These combined data enabled usto establish a new genus of marine gregarines that providesthe cellular identity of a clade of SSU rDNA environmentalsequences isolated from various marine environmentsaround the globe.

Materials and methods

Collection of organisms

The capitellid polychaete, Notomastus tenuis Moore, 1909,was collected at low tide from Boundary Bay, Tsawwassen(Vancouver), British Columbia, Canada in August 2011. Asecond capitellid polychaete, Heteromastus filiformisClaparède, 1864, was collected from the rocky intertidalarea at Jericho Beach, Vancouver, British Columbia,Canada, in September 2011. No specific permits were re-quired for the collection of worms in these field sites. Tidelevels were estimated and acquired through Fisheries andOceans Canada. Host material was transported and kept inchilled seawater prior to dissection. All dissections werecompleted within 24 h of collection. No fixatives were usedeither during dissections or while taking photographs ofthe trophozoites. Three different morphotypes of grega-rine trophozoites (Polyplicarium lacrimae n. gen. et sp.,P. curvarae n. gen. et sp., and P. translucidae n. gen. etsp.) were collected from the intestines of N. tenuis; onedistinct morphotype of a gregarine trophozoite (P. cit-rusae n. gen. et sp.) was collected from the intestines ofH. filiformis.

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Light microscopy and single-cell DNA extraction,amplification and sequencing

Hand-drawn glass pipettes were used to collect individualtrophozoites representing four distinct morphotypes, usingan inverted microscope (Zeiss Axiovert 200; Carl-Zeiss,Göttingen, Germany). Four single-cell (SC) isolates werecollected from each of the four distinct morphotypes (a totalof 16 SC isolates) and prepared for light microscopy andDNA extraction. The SC isolates were washed three times(until clean) in chilled, autoclaved seawater and photo-graphed either on glass slides with a Leica DC 500 colorcamera connected to a Zeiss Axioplan 2 microscope (Carl-Zeiss) or on well-slides with a PixeLink Megapixel colordigital camera (PL-A662-KIT; Ottawa, Canada) connectedto an inverted Zeiss Axiovert 200 microscope (Carl-Zeiss).Each of the SC isolates was then placed in a 1.5-mlEppendorf tube containing cell lysis buffer. GenomicDNA was extracted with the standard protocol providedby the MasterPure complete DNA & RNA purificationkit (Epicentre Biotechnologies, Madison, WI, USA).However, the final elution step was lowered to 4 μlwith the goal of concentrating extracted DNA prior toPCR amplification.

Sixteen novel SSU rDNA sequences were generated bynested PCR with primers specific for the gregarine parasitespecies (Table 1). Initially, outside primers PF1 and SSUR4(Leander et al. 2003) were used in a 25-μl PCR reactionwith EconoTaq 2X Master Mix (Lucigen, Middleton, WI,USA). The following program was used on the thermocyclerfor the initial amplification: initial denaturation at 94 °C for2:00 min; 35 cycles of denature at 94 °C for 0:30 s, anneal at52 °C for 0:30 s, extension at 72 °C for 1:50 m., finalextension 72 °C 9:00 m. Subsequently, a pair of internalprimers, F1 and R2 (Table 1), were used in a nested PCRwith 1 μl of template DNA generated from the first PCRreaction in order to amplify a 1,000-bp region of the SSUrRNA gene using the following program on a thermocycler:initial denaturation for 94 °C for 2:00 min; 25 cycles ofdenature at 94 °C for 0:30 s., anneal at 51 °C for 0:30 s.,

extension at 72 °C for 1:20 min; final extension at 72 °C for9:00 min.

From the initial sequences, specific primers were thendesigned and paired with outside (universal eukaryotic)primers (e.g., PF1-PlacrimaeR and PlacrimaeF-SSUR4) insemi-nested PCR reactions in order to attain the final SSUrDNA sequences (1,650–1,800 bp) (Table 1). All PCRproducts were separated on agarose gels and isolated usingthe UltraClean15 DNA Purification Kit (MO BIO,Laboratories, Carlsbad, CA, USA). All sequencing reactionswere performed using ABI big dye reaction mix with ap-propriate primers (Table 1). Novel sequences were initiallyidentified using the National Center for BiotechnologyInformation’s (NCBI) BLAST tool and confirmed with mo-lecular phylogenetic analyses. All unique sequences gener-ated in this study were deposited in GenBank (Accessionnumbers JX535336–JX535351).

Scanning electron microscopy

Between 20 and 65 individual trophozoites representingeach morphotype were pooled in 2 % glutaraldehyde inseawater on ice. A 10-μl polycarbonate membrane filterwas placed within a Swinnex filter holder (Millipore,Billerica, MA, USA). Trophozoites were then collected witha hand-drawn glass pipette and placed in the filter holder,which was then placed in a small beaker (4 cm diam. and5 cm tall) that was filled with 2 % glutaraldehyde in seawa-ter. Ten drops of 1 % OsO4 were added to the opening of thefilter holder, and the samples were post-fixed on ice for30 min. A syringe was used to slowly run distilled waterover all samples. A graded series of ethanol washes (30, 50,75, 85, 95, and 100 %) was then used to dehydrate the fixedcells using the syringe system. Following dehydration, thepolycarbonate membrane filters containing the trophozoiteswere transferred from the Swinnex filter holders into analuminum basket submerged in 100 % ethanol in prepara-tion for critical point drying with CO2. The dried polycar-bonate membrane filters containing the trophozoites weremounted on aluminum stubs, sputter coated with 5 nm gold

Table 1 Primers designed inthis study to amplify smallsubunit rDNA sequences; theannealing regions refer to one ofthe sequences derived fromPolyplicarium lacrimae n. gen.et sp. (GenBank accession no.JX535336)

Primer name Direction Sequence 5'–3' Annealing region

F1 Forward 5'-GATTAAGCCATGCATGTCTAAG-3' 47 to 70

P. lacrimae F Forward 5'-CGTTTCTACGATTATCAATTGG-3' 486 to 508

P. curvarae F Forward 5'-CGTTTCTATGAGTACCCATTGG-3' 486 to 508

P. translucidae F Forward 5'-CGTTTCTACGATTACCCATTGG-3' 486 to 508

P. citrusae F Forward 5'-CTTTCTACGAGTACCAATTGG-3' 486 to 508

R1 Reverse 5'-CGGTGTGTACAAACGGCAGGGAC-3' 1762 to 1740

P. lacrimae R Reverse 5'-CTGACAGGGCCGAGGTCCTATCG-3' 671 to 648

P. curvarae R Reverse 5'-CGGATAAGACGGAAGTCCTATCG-3' 671 to 648

P. translucidae R Reverse 5'-GGGATAGGACGGAAGTCCTATAG-3' 671 to 648

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and viewed under a Hitachi S4700 scanning electron micro-scope (Nissei Sangyo America, Pleasanton, CA, USA).Some SEM data were presented on a black backgroundusing Adobe Photoshop 6.0 (Adobe Systems, San Jose,CA, USA).

Phylogenetic analyses of molecular sequences

Two separate phylogenetic analyses were conducted in thisstudy. A comprehensive 79-taxon dataset contained a repre-sentative SSU rDNA sequence from each of the four novelmorphotypes described here: five closely related environ-mental DNA sequences, three dinoflagellate sequences(outgroup), and 67 sequences representing major clades ofgregarines and other apicomplexans. The 79-taxon align-ment was visually fine-tuned using MacClade 4 (Maddisonand Maddison 2000); gaps and ambiguously aligned regionswere excluded resulting in 1,007 unambiguously alignedsites. JModeltest (Guindon and Gascuel 2003; Posada andCrandall 1998) selected a GTR+I+Γ model of evolutionunder AIC and AICc (proportion of invariable sites00.1280, gamma shape00.5250). Garli-GUI (Zwickl 2006)was used to generate a maximum likelihood (ML) tree, andML bootstrap analysis (100 pseudoreplicates, one heuristicsearch per pseudoreplicate).

Bayesian posterior probabilities were calculated for thelarger dataset using the programMrBayes 3.1.2 (Huelsenbeckand Ronquist 2001; Ronquist and Huelsenbeck 2003). We setour program for four Monte Carlo Markov Chains startingfrom a random tree (MCMC; default temperature00.2), agamma distribution and stop rule of 0.01 (i.e. when the aver-age split deviation fell below 0.01, the program would termi-nate). A sum of 5,000,000 generations was calculated. Treeswere sampled every 100 generations, with a prior burn-in of500,000 generations. Burn-in was confirmed manually, and amajority-rule consensus tree was constructed. Posterior prob-abilities correspond to the frequency at which a given node isfound in the post-burn-in trees.

A more restricted phylogenetic analysis focused on theinterrelationships between the 16 SSU rDNA sequencesgenerated from four different single-cell isolates from eachof the four different morphotypes described here; the align-ment also contained the five closely related environmentalDNA sequences identified in GenBank. This 21-taxonalignment was visually fine-tunedwithMacClade 4, excludedgaps and ambiguous sites, and contained 1,407 sites. Anunrooted ML tree and ML Bootstrap percent values werecalculated using Garli-GUI under a GTR+I+Γ model ofevolution selected by JModeltest (proportion of invariablesites00.4970, gamma shape00.5830). Like the larger 79-taxon dataset, posterior probabilities were calculated usingMrBayes 3.1.2. using the same criteria described previously.A sum of 700,000 generations was calculated with a prior

burn-in of 70,000 generations. PAUP 4.0 (Swofford 1999)was used to calculate percent differences between the 16novel SSU rDNA sequences generated in this study.

Results

Morphological traits of the four new species

Polyplicarium lacrimae n. gen et sp. The trophozoites wereteardrop-shaped (Fig. 1a–e). The anterior end of the cell wasbulbous, having an average width of 54 μm (range 38–66 μm, n042) at its widest part. The average length of thecell was 197 μm (range 183–207 μm, n042). Cellsappeared dark-brown from large amounts of amylopectin.The nucleus was circular to ovoid (17–22 μm×15–21 μm,n017) and located in the central part of the bulbous anteriorof the cell (Fig.1a–e). The anterior end tapered slightlytoward a blunt and otherwise inconspicuous mucron. Theposterior end tapered to a point (Fig. 1a–e). Longitudinalepicytic folds covered the surface of the cell with a densityof 4–5/μm (Fig. 1f, g). Gliding motility was present. Adistinct region of shallow epicytic folds was observed onthe surface of cells examined under SEM (Fig. 1g, h). Thisregion was observed in two out of the six samples recoveredfor viewing.

Polyplicarium curvarae n. gen. et sp. The trophozoites wereslightly curved, 156 μm long (range 98–167 μm, n047) and42 μm wide (range 34–53 μm, n047) (Fig. 2a–e). Thenucleus was ovoid (16–21 μm×10–12 μm, n021) andlocated in the posterior region of the cell. A conspicuousmucron was visible in cells under light microscopy(Fig. 2d); otherwise, the mucron appeared flat and incon-spicuous (Fig. 2a–c). Cells appeared brown from the accu-mulation of amylopectin. SEMs demonstrated longitudinalepicytic folds covering the cell surface with a density of 4–5/μm (Fig. 2f). The posterior end of the trophozoites taperedslightly to a blunt and slightly compressed end (Fig. 2a–e).The trophozoites were capable of gliding motility. Thesurface of the cell contained a distinct region of 12–17shallow epicytic folds that were 2–3 μm wide and tallerthan the other epicytic folds (Fig. 2h). This pattern ofepicytic folds was observed in 11 out of 20 cells observedunder SEM.

Polyplicarium translucidae n. gen. et sp. Trophozoites were163 μm long (range 112–183 μm, n059) and 27 μm wide(range 25–32 μm, n059) (Fig. 3a–e). The nucleus wascircular to ovoid (12–15 μm×7–13 μm) and located in themiddle to posterior end of the cell. The trophozoites werecapable of gliding motility. The cells appeared translucentunder light microscopy, having a low accumulation of

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amylopectin granules. The posterior end was slightly ta-pered and sometimes tapered to a nipple-like point(Fig. 3a–e). Longitudinal epicytic folds covering the surfacehad a density of 4–5/μm (Fig. 3f, g). A distinct region of10–13 shallow epicytic folds was observed on the surface ofthe cell (Fig. 3f, g). This pattern of epicytic folds wasobserved in 17 out of 30 cells observed under SEM.

Polyplicarium citrusae n. gen et sp. The trophozoites wereextremely flat, ovoid to lemon-shaped, 47 μm long (range39–53, n049) and 32 μm wide (range 28–42, n049)(Fig. 4a–d). The circular to ovoid nucleus (4–7 μm×4–8 μm, n015) was located in the central part of the cell. Cellsappeared translucent to light-brown, depending on theamount of amylopectin granules present within the cell.Both the anterior and posterior ends were slightly tapered.The trophozoites were capable of gliding motility. Theanterior end was differentiated from the posterior end

mainly by observing the direction of “forward” glidingmotility. Syzygy was side-to-side (Fig. 4e). The cell sur-face was covered with longitudinal epicytic folds with adensity of 4/μm (Fig. 4e, f). No distinct region of shallowepicytic folds was present on the 32 cells observed underSEM.

Molecular phylogenetic analyses of SSU rDNA sequences

Our phylogenetic analysis of the 79-taxon dataset recovereda clade of apicomplexans with moderate support. Our anal-yses also recovered groups of coccidians, piroplasmids,rhytidocystids, cryptosporidians, and terrestrial gregarines,ranging in support from moderate to robust (Fig. 5). Fourmajor subgroups of marine gregarines were recovered in thisanalysis: crustacean gregarines, lecudinids clade I, lecudi-nids clade II plus urosporids, and the novel clade establishedhere from capitellid hosts (Fig. 5). Species of Selenidium

Fig. 1 Light micrographs (LM) and scanning electron micrographs(SEM) showing the trophozoite morphology and surface features ofPolyplicarium lacrimae n. gen. et sp. a–d The LMs were taken ofindividual cells on a well-slide just before they were removed for DNAextraction and single-cell PCR. Individual trophozoites with a bluntmucron (arrow) and bulbous anterior containing a centrally locatednucleus (N). The posterior region tapers to a point. e LM showing thegeneral morphology of P. lacrimae, the inconspicuous mucron (arrow),

centrally located nucleus (N), and a pointed posterior end. f High-magnification SEM of the cell surface of P. lacrimae showing theepicytic folds (arrowhead). g SEM showing the general morphology,the inconspicuous mucron (arrow), the pointed posterior end of thecell, and the distinct region of wider folds on a trophozoite (triple-arrowhead). h High-magnification SEM of the distinct region of widerfolds (triple-arrowhead) on trophozoites. Scale bars (a–d) 20.0 μm,(e) 25 μm, F(f) 1 μm, (g) 15 μm, (h) 1.5 μm

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(i.e., archigregarines) branched from the unresolved api-complexan backbone as three separate lineages. The fournovel sequences representing P. lacrimae, P. curvarae, P.translucidae, and P. citrusae formed a well-supported cladewith five environmental sequences of previously unknownorigin within the Apicomplexa (AY179976, EF100216,EF100199, AB275013, and AY179975) (Fig. 5).

Molecular phylogenetic analyses with the 21-taxon datasetcontaining five environmental sequences and the 16 new SSUrDNA sequences (i.e., four from single-cell isolates of each ofthe four species in this study) are shown in Fig. 6. The analysisrecovered four distinct, well-supported clades that representedthe single-cell isolates from each species described in thisstudy. A fifth clade consisted of four environmental sequences(AB275013, EF100199, EF100216, and AY179976), andenvironmental sequence AY179975 branched as the sisterlineage to P. citrusae with strong statistical support (Fig. 6).

Intraspecific variation of the four SSU rDNA sequencesgenerated from each of the four species ranged from 0.98 to2.12 % (P. lacrimae), 0.22 to 1.46 % (P. curvarae), 0.68 to1.73 % (P. translucidae), and 0.82 to 1.35 % (P. citrusae)(Table 2). Interspecific variation between isolates of P.

lacrimae P. curvarae, P. translucidae, and P. citrusaeranged from 7.42 to 14.98 % (Table 2).

Formal taxonomic descriptions

Apicomplexa Levine, 1970Gregarinea Bütschli, 1882, stat. nov. Grassé, 1953Eugregarinorida Léger, 1900

Polyplicarium n. gen. Wakeman and Leander

Description Ovoid to elongate trophozoites with a bluntmucron. The posterior end is either blunt or tapers to apoint. Longitudinal epicytic folds with density of 4–5/μm;most trophozoites also have a distinct region of wider,shallower epicytic folds. Gliding motility. The genus name,Polyplicarium, is latin, translates to “many folds”, and refersto the high density of epicytic folds on the surface of thetrophozoite stages.

Type species Polyplicarium lacrimae Wakeman andLeander.

Fig. 2 Light micrographs (LM) and scanning electron micrographs(SEM) showing the trophozoite morphology and surface features ofPolyplicarium curvarae n. gen. et sp. a–d The LMs were taken ofindividual cells on a well-slide just before they were removed for DNAextraction and single-cell PCR. General morphology of cup-like (a–b)or projected (d) mucrons (arrow). The nucleus (N) is located in theposterior or middle-posterior part of the cell. The posterior end tapersslightly to a blunt end. e LM showing the general morphology of atrophozoite with the mucron (arrow), nucleus (N), and a distinct fold

(triple arrowhead) in the center of the trophozoite. f High-magnification SEM showing the mucron (arrow) and dense epicyticfolds (arrowhead) on surface of the cell. g SEM of a trophozoiteattached to host gut (HG) material. The interface between the hostgut and trophozoite is marked by an arrow. The posterior region of thecell is blunt and slightly compressed. h SEM showing the generalmorphology of the cell, the mucron (arrow), and a blunt posterior end.A distinct region of wider folds (triple-arrowhead) was observed on thecell surface. Scale bars (a–d) 30 μm, (e) 30 μm, (f) 3.5 μm, g, h) 10 μm

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Polyplicarium lacrimae n. sp. Wakeman and Leander

Description Trophozoites teardrop-shaped with a bulbousmucron. Average length and width, at the widest part, is197 μm and 54 μm, respectively. Cells dark-brown. Theposterior end tapered to a point; anterior end tapered slightlytoward a blunt, inconspicuous mucron. Nucleus is circularto ovoid (17–22 μm×15–21 μm) and located in the anterioror central part of the cell. Gliding motility. Longitudinalepicytic folds with a density of 4–5/μm. A distinct regionof wider, shallower epicytic folds may be present on thesurface of trophozoite stages.

DNA sequence SSU rRNA gene sequence (GenBankJX535336).

Type locality Boundary Bay (49°00′54.88″N, 123°02′12.72″W), Tsawwassen (Vancouver), British Columbia, Canada.Host in sand; upper intertidal; 0.30 m above mean sea level.

Type habitat Marine.

Type host Notomastus tenuis Moore, 1909 (Annelida,Polychaeta, Scolecida, Capitellidae).

Location in host Intestinal lumen.

Iconotype Figure 1g.

Hapantotype Parasites on gold sputter-coated SEM stubshave been deposited in the Beaty Biodiversity Museum

Fig. 3 Differential interference contrast light micrographs (LM) andscanning electron micrographs (SEM) showing the trophozoite mor-phology and surface features of Polyplicarium translucidae n. gen. etsp. a–d The LMs were taken of individual cells on a well-slide justbefore they were removed for DNA extraction and single-cell PCR.Trophozoites have a posterior nucleus (N), a rounded mucron (arrow),and a posterior end that tapers slightly to a nipple-like point (P) (b–c). e

LM showing the general cell morphology, the mucron (arrow), theposterior nucleus (N), and a region of wider folds (triple arrowhead). fHigh-magnification SEM showing the dense epicytic folds (arrowhead)and a region of wider folds (triple arrowhead) on the cell surface. h SEMshowing the mucron (arrow), the nipple-like posterior end of the cell, andthe distinct region of wider folds (triple-arrowhead). Scale bars (a–e)25 μm, (f) 2.0 μm, (g) 10 μm

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(Marine Invertebrate Collection) at the University of BritishColumbia, Vancouver, Canada. Museum Code – MI-PR117.

Etymology The species name, lacrimae, stems from Latinmeaning “tear” and refers to the “teardrop-shape” of thetrophozoite stage.

Polyplicarium curvarae n. sp., Wakeman and Leander

Description Trophozoites slightly curved with an averagelength and width of 156 μm and 42 μm, respectively. Cellsbrown. The posterior end of trophozoite slightly tapered andcompressed. Ovoid nucleus located in the posterior regionof the cell. Mucron usually flat and inconspicuous butsometimes pointed. Gliding motility. Longitudinal epicyticfolds with a density of 4–5/μm. A distinct region of 15–17wider and shallower epicytic folds present on the trophozoitesurface.


Type locality Boundary Bay (49°00′54.88″N, 123°02′12.72″W), Tsawwassen (Vancouver), British Columbia, Canada.Host in sand; upper intertidal; 0.30 m above mean sea level.




Iconotype Figure 2h.

Hapantotype Parasites on gold sputter-coated SEM stubshave been deposited in the Beaty Biodiversity Museum(Marine Invertebrate Collection) at the University of BritishColumbia, Vancouver, Canada. Museum Code – MI-PR118.

Etymology The species name, curvarae, stems from Latinmeaning “curved” and refers to the curved cell shape of thetrophozoites stage.

Polyplicarium translucidae n. sp., Wakeman and Leander

Description Elongated trophozoites 163 μm long and27 μm wide on average. The circular to ovoid nucleus waslocated in the posterior half of the cell. The posterior endtapered to a nipple-like tip. Gliding motility. Trophozoiteswith an inconspicuous mucron and distinctively translucentunder light microscopy. Longitudinal epicytic folds with adensity of 4–5/μm over the trophozoite surface. A distinctswelling of 6–10 wider epicytic folds was present on one sideof the trophozoite surface.

Fig. 4 Differential interferencecontrast light micrographs(LM) and scanning electronmicrographs (SEM) showingthe trophozoite morphologyand surface features ofPolyplicarium citrusae n. gen.et sp. a–d The LMs were takenof individual cells just beforethey were removed for DNAextraction and single-cell PCR.Individual trophozoites wereextremely flattened and had acentrally located nucleus (N).The mucron (arrow) wasidentified based on the forwarddirection of movement. e SEMshowing two gamonts (G1 andG2) in side-to-side syzygy andepicytic folds running along thelongitudinal axis of the cell. fSEM of a single trophozoiteshowing the epicytic folds(arrowhead). The anterior andposterior ends of these cells wasdifficult to distinguish underSEM. Scale bars (a–e) 10 μm,(f) 5 μm

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Type locality Boundary Bay (49°00′54.88″N, 123°02′12.72″W), Tsawwassen (Vancouver), British Columbia, Canada.Host in sand; upper intertidal; 0.30 m above mean sealevel.




Fig. 5 Maximum likelihood(ML) tree based on 1,007unambiguously aligned sitesfrom 79 SSU rDNA sequencesusing the GTR+I+Γ substitutionmodel (−ln L0168,00.87694,gamma shape00.5250, propor-tion of invariable sites00.1280).Bootstrap supports are given atthe top of braches, and Bayesianposterior probabilities are givenat the bottom. Black dots onbranches represent bootstrapsupport values and Bayesianposterior probability 95/0.99 orgreater. Bootstrap and Bayesianvalues less than 55 and 0.95,respectively, were not addedto this tree. Representativesequences from the four novelspecies described in this studyare highlighted in black boxes

Mar Biodiv (2013) 43:133–147 141

Iconotype Figure 3g.

Hapantotype Parasites on gold sputter-coated SEM stubshave been deposited in the Beaty Biodiversity Museum(Marine Invertebrate Collection) at the University ofBritish Columbia, Vancouver, Canada. Museum Code –MI-PR119.

Etymology The species name, translucidae, stems fromLatin meaning “transparent”, and refers to the “see-through” quality of the trophozoites stages of thisspecies.

Polyplicarium citrusae n. sp., Wakeman and Leander

Description Trophozoites of P. citrusae extremely flattenedand lemon-shaped with an average length and width of 47and 32 μm, respectively. Trophozoites translucent to light-brown under light microscopy. Circular nucleus positionedin the center of trophozoites. Gliding motility. The anteriormucron region was inconspicuous and difficult to distin-guish from the posterior end, without observing the direc-tion of gliding motility. The cell surface was coveredlongitudinal epicytic folds with a density of 4/μm. Syzygyside-to-side.

Fig. 6 Unrooted maximumlikelihood (ML) tree of foursingle-cell isolates from eachof the four novel species ofPolyplicarium n. gen. describedin this study, as well as fiveclosely related environmentalsequences. This tree is basedon 1,407 unambiguouslyaligned sites from 21 SSUrDNA sequences using theGTR+I+Γ substitution model(−ln L016,800.87694, gammashape00.5830, proportion ofinvariable sites00.4970).Bootstrap supports are givenat the top of branches, andBayesian posterior probabilitiesare given at the bottom. Blackdots on branches representbootstrap support values andBayesian posterior probability95/0.99 or greater. Bootstrapand Bayesian values less than55 and 0.95, respectively, werenot added to this tree

Table 2 Summary of intraspecific divergences (along the diagonal)and interspecific divergences of small subunit rDNA sequencesgenerated from four isolates of each of the four species of

Polyplicarium n. gen. described in this study; percent divergencesare based on comparisons of 1,678 nucleotides.

Polyplicarium lacrimaen. gen. et sp.

P. curvaraen. gen. et sp.

P. translucidaen. gen. et sp.

P. citrusaen. gen. et sp.

Polyplicarium lacrimae n. gen. et sp. 0.98–2.12 % – – –

P. curvarae n. gen. et sp. 10.67–12.53 % 0.22–1.46 % – –

P. translucidae n. gen. et sp. 9.39–11.10 % 7.42–9.02 % 0.68–1.73 % –

P. citrusae n. gen. et sp. 13.21–14.98 % 8.58–10.07 % 10.17–12.09 % 0.82–1.35 %

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Type locality Jericho Beach (49°16′24.39″N, 123°11′07.18″W), Point Grey (Vancouver), British Columbia, Canada.Host in black sediment; mid-low intertidal; 0.80 m belowmean sea level.


Type host Heteromastus filiformis Eisig, 1887 (Annalida,Polychaeta, Scolecida, Capitellidae).


Iconotype Figure 4f.

Hapantotype Parasites on gold sputter-coated SEM stubshave been deposited in the Beaty Biodiversity Museum(Marine Invertebrate Collection) at the University ofBritish Columbia, Vancouver, Canada. Museum Code –MI-PR120.

Etymology The species name, citrusae, stems from Latinmeaning “citrus”, and refers to the general “lemon-shape”of the trophozoites and gamonts.

Discussion

Species of marine gregarines have been established using awide variety of criteria, including hosts affinity, geographical

distribution, detailed morphological dimensions of differentlife history stages, ultrastructural patterns on the surface oftrophozoites, and, more recently, SSU rDNA sequence vari-ability (Leander et al. 2003; Rueckert and Leander 2008;Rueckert et al. 2010, 2011a, b). Traditionally, new specieswere justified mainly on just one of these criteria (e.g., detailedmorphological dimensions of different life history stages).More recent studies suggest that one criterion alone is inade-quate to convincingly delimit different species of marinegregarines from one another (Rueckert et al. 2011a, b). Forinstance, the SSU rDNA sequences from very different mor-photypes of Lecudina polymorpha were over 99 % identical(Leander et al. 2003; Rueckert et al. 2010). By contrast, theSSU rDNA sequences from morphologically similar gregar-ines isolated from two different species of nemerteans differedby 14.1 %, demonstrating two different species of Difficilinathat were correlated with two different host species (Rueckertet al. 2010). A similar study described two different species ofLankesteria isolated from two separate species of tunicates(Rueckert and Leander 2008). Because the SSU rDNAsequences from these two species differed only by 2.1–3.1 %, the separation of the two species was also based onmorphological variation (e.g., L. chelyosomae was over 10times larger than L. cystodytae), different host affinities, andthe fact that multiple isolates from each species clustered intotwo distinct clades (Rueckert and Leander 2008). Anotherstudy addressed the SSU rDNA sequence variation in severaldifferent morphotypes of Lecudina cf. tuzetae isolated fromdifferent hosts collected in two different geographicalregions (Rueckert et al. 2011b). The range of variation inthese sequences was 0.0–3.9 %; however, the sequencesdid not cluster into clades according to morphotype,

Table 3 Comparative morphology of the four new species of Polyplicarium n. gen. described in this study

Polyplicarium lacrimae n.gen. et sp. (type species)

P. curvarae n. gen. et sp. P. translucidae n.gen. et sp.

P. citrusae n. gen.et sp.

Host Notomastus tenuis Notomastus tenuis Notomastus tenuis Heteromastus filiformis

Host tissue Intestines Intestines Intestines Intestines

Locality E. Pacific E. Pacific E. Pacific E. Pacific

Trophozoite shape Elongate, bulbous anterior,posterior end tapered to point

Elongate, cylindrical, posteriorend slightly compressed

Elongate, compressed,posterior blunt

Round to ovoid,highly flattened

Tophozoite size

L × W, μm 183–207×38–66 98–167×34–53 112–183×25–32 39–53×28–42

Nucleus shape Ovoid Ovoid Ovoid Ovoid

Nucleus size

L × W, μm 17–22×15–21 16×21×10–12 12–15×7–13 4–7×4–8

Position of nucleus Middle anterior Middle posterior Middle posterior Middle

Motility Gliding motility Gliding motility Gliding motility Gliding motility

Density of epicytic folds 4–5/μm 4–5/μm 4–5/μm 4/μm

Shape of mucron Simple, blunt Simple, blunt Simple, blunt Simple, blunt

Region with alternative fold pattern Present Present Present Absent

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location or host, suggesting that the degree of variationfound in this study was intraspecific. Taken together, thesestudies illustrate the importance of considering multiplecriteria to justify the establishment of new species andgenera (Leander et al. 2003; Rueckert et al. 2010, 2011a,b). Arguably, the most pragmatic approach for describingnew species of gregarines is to consider host affinity,morphological features of the most conspicuous life historystage (usually trophozoites), and a widely sampled molecu-lar marker with sufficient interspecific variation (e.g., SSUrDNA sequences) using a single-cell PCR approach(Rueckert et al. 2011b).

A recently published list of “6 fundamental principles”for understanding species boundaries is a great testamentto the shortcomings of delimiting gregarine species basedon detailed morphometric data alone (Clopton 2012).According to Clopton (2012), gregarine systematics mustinclude: (1) an ability to sort out mature life stages fromimmature life stages and all developmental stages inbetween; (2) an ability to observe all lifecyle stages; (3)an ability to sort out sexually dimorphic gamonts, ifpresent; (4) large sample sizes of morphometric detailsthat reflect the entire population; (5) measurements ofmorphology that are free of osmotic and other prepara-tion artifacts; and (6) comparable and detailed morpho-metric data of all other closely related species. Thesegoals not only position idealism above realism but reflecta pre-DNA worldview extrapolated from research ongregarines living in cockroaches and other insects. Inpractice, marine gregarines are encountered only astrophozoites and often in very low numbers (fewer than10 specimens in an individual host) within a small per-centage of individual hosts, which are also difficult toencounter and collect (e.g., the hosts might have beencollected on a research cruise that was a one-time oppor-tunity). More importantly, the challenges emphasizedwithin the principles listed by Clopton (2012) are need-less and overcome by efficient and pragmatic DNA-basedapproaches to systematics, and the literature is rich inexcellent studies that demonstrate this in a wide varietyof organisms and a broad range of contexts (e.g., biogeo-graphy, cryptic lifecycle stages, and cryptic species).Although reciprocal reinforcement of molecular data andother traits (e.g., trophozoite features and host affilia-tions) provide the most compelling arguments for speciesdiscrimination, a viewpoint that insists on an absolute setof morphometric details in gregarine systematics simplystifles the enterprise and provides a stark counter-example for why DNA-based approaches to biogeogra-phy, the delimitation of species, and phylogenetic recon-struction have become so predominant in advancing ourunderstanding of biodiversity, especially within the con-text of protists.

Justification for establishing the new genus and species

Nearly 1,700 species of gregarines have been formally de-scribed, often with only very limited morphological infor-mation; only a tiny fraction of these have been examinedwith molecular phylogenetic data (Leander 2008; Perkins etal. 2002; Levine 1971, 1976, 1977a, b, 1979). Molecularmarkers like SSU rDNA sequences that have been obtainedfrom manually isolated trophozoites have been helpful inour understanding of gregarine species boundaries and phy-logenetic relationships (Leander 2007; Leander et al. 2006;Rueckert and Leander 2008, 2009, 2010; Rueckert et al.2010, 2011a, b; Wakeman and Leander 2012). Analyses ofSSU rDNA sequences derived directly from known specieshave also been able to establish the cellular identities ofambiguous environmental DNA sequences that have accu-mulated from PCR surveys of biodiversity (Cavalier-Smith2004; Rueckert et al. 2011a, b). Nonetheless, the absence ofmolecular data from the vast majority of described gregarinespecies severely constrains the comparative power needed toplace newly discovered species within the context of knowngregarine species.

In this study, we established four novel species withinPolyplicarium n. gen. using comparative morphology andSSU rDNA sequences from four different single-cell iso-lates of each of the four different species. The trophozoitemorphology of Polyplicarium species was most similar tothe following genera within the Lecudinidae: Hyperidion,Ancora, Ulvina, Zygosoma, and Lecudina. The trophozoitesin all of these genera have “simple mucrons” but differ fromone another in several ways (Lee et al. 2000; Perkins et al.2002). For instance, the trophozoites of Hyperidion are 175–350 μm long, contractile, have longitudinal folds with adensity of about 2–3/μm, and have a textured projectionfrom the mucron (Lee et al. 2000; Mackinnon and Ray1931). The trophozoites of Polyplicarium are shorter andalmost twice as wide and have longitudinal folds with adensity of 4–5/μm. The trophozoites of Ancora have two–three lateral processes running from the mucron towards theposterior end (Hoshide 1998; Levine 1977a, b). LikePolyplicarium, two species of Ancora (A. sagitatta and A.lutzi) were isolated from a capitellid polychaete (Capitellacapitata); however, Polyplicarium species do not have thelateral processes that define Ancora (Hasselmann 1918).The morphology of Polyplicarium is also different fromUlvina and Zygosoma (Lee et al. 2000; Mackinnon andRay 1931). Unlike Polyplicarium, the trophozoites ofUlvina have an incomplete septum dividing the cell into a“pseudoprotomerite” and “pseudodeutomerite”, and thetrophozoites/gamonts of Zygosoma are covered withnipple-like projections (Lee et al. 2000). Lecudina is thelargest and most widely studied genus within theLecudinidae (Clausen 1993; Hasselmann 1918; Leander et

144 Mar Biodiv (2013) 43:133–147

al. 2003; Lee et al. 2000; Levine 1976). Several species ofLecudina, including the type species L. pellucida, havetrophozoites with a density of epicytic folds in the rangeof 2–3/μm (Leander et al. 2003; Rueckert et al. 2011a, b;Vivier 1968), which is about half the density of folds on thesurface of trophozoites in Polyplicarium. Moreover, all spe-cies of Lecudina that have been examined with SSU rDNAsequences do not cluster with or within the Polyplicariumclade in molecular phylogenetic analyses.

Aside from the molecular phylogenetic data, the mainfeatures that delimit the trophozoites of Polyplicarium fromother gregarine genera within the Lecudinidae are longitu-dinal epicytic folds with density of 4–5/μm, a distinct regionof wider epicytic folds (usually), and host affiliation. Thefour different species of Polyplicarium n. gen. describedhere can be distinguished from one another based on detailsof trophozoites morphology and SSU rDNA sequence var-iation (Tables 2, 3). Unlike the other species, the tropho-zoites of P. lacrimae have a bulging region in the anterior-middle region of the cell, a centrally located nucleus, and aposterior end that tapers to a distinct point. A previouslydescribed species by Lankester (1866), namely Lecudinaeunicae, had a similar cell shape (boulbous anterior and apointed posterior) and size (254 μm) to that of P. lacrimae.However, the species described by Lankester (1866) wasisolated from a different host, Eunice harassii, and theoriginal drawing of the trophozoite of L. eunicae shows adistinct bulbous anterior region that is about half the cell’stotal length. In contrast, the anterior region of the tropho-zoites of P. lacrimae appear to be about 2/3 the total lengthof the cell. The trophozoite stage and host affinity of L.eunicae is most similar to that of Trichotokara eunicae,recently described by Rueckert et al. (2012), and most likelyrepresents a close relative those gregarines isolated fromEunicid polychaetes (Rueckert et al. 2012).

The trophozoites of P. curvarae had a posteriorly posi-tioned nucleus and were distinctly curved and cylindrical,compared to P. lacrimae, and P. translucidae (Table 3). Thetrophozoites of P. translucidae lacked a large number ofamylopectin granules within the cytoplasm, giving this spe-cies a characteristic transparent appearance under light mi-croscopy. In contrast to the other species of Polyplicarium, P.citrusae is highly flattened and relatively small with an aver-age length and width of only 53 and 29 μm, respectively. Thetrophozoites of P. citrusae were also isolated from the intes-tines of different capitellid host, namely Heteromastus filifor-mis. The general outline shape of P. citrusae was distinctlyovoid, reminiscent of a lemon (Table 3).

In contrast to P. citrusae, the surface of the trophozoitesin P. lacrimae, P. curvarae, and P. translucidae had adistinct region of wider and shallower epicytic folds.Although the functional significance of this particular regionof epicytic folds in these three species is uncertain, it is

plausible that the folds facilitate the acquisition of nutrientsby expanding and contracting, thereby moving contents inthe host gut around the cell. The consistent presence of thisregion of shallow folds in repeated observations of threedifferent species, each prepared multiple times, minimizedthe chance that this distinct feature reflects a preparationartifact.

Our molecular phylogenetic analyses of SSU rDNAgrouped the four single-cell isolates from each of the fourspecies into four separate and corresponding clades (Fig. 6).Intraspecific variation of the SSU rDNA sequence withineach clade was low, ranging from 0.22 to 2.12 % (Table 2).Interspecific variation between the four clades was relativelyhigh, ranging from 7.42 to 14.98 % (Table 2). The morpho-logical features of the trophozoites combined with the phy-logenetic pattern of SSU rDNA sequence variation providedstrong evidence for the delimitation of all four species ofPolyplicarium from one another.

Environmental SSU rDNA sequences and the Polyplicariumclade

The four species of Polyplicarium that we described heregrouped strongly with five SSU rDNA environmentalsequences of similar branch length that were retrieved fromGenBank. Environmental sequences AY179975 andAY179976 were generated from a PCR survey of sedimentin a salt marsh near Cape Cod, Massachusetts, USA; envi-ronmental sequences EF100199 and EF100216 generatedfrom a PCR survey of sediment from a marine tidal flat offthe coast of Greenland (Stoeck and Epstein 2003); environ-mental sequence AB275013 was generated from sedimentfrom a deep sea methane cold seep near Sagami Bay, Japan(Takishita et al. 2007a, b). Until now, the cellular identity ofthese environmental sequences was either considered uncer-tain within gregarine apicomplexans (Cavalier-Smith 2004;Leander 2007; Leander et al. 2006; Rueckert and Leander2008, 2009, 2010; Rueckert et al. 2010, 2011a, b) or entirelymisinterpreted (e.g., novel jacobid-like sequences) (López-García et al. 2007; Stoeck and Epstein 2003). Nonetheless,the vastly different geographical locations from which theenvironmental DNA sequences were generated indicate thatthe Polyplicarium clade has a global distribution and that weare at an early stage of understanding the total compositionof this clade.

Concluding remarks

This study represents the first molecular phylogenetic datagathered from gregarines isolated from capitellid poly-chaetes. The combination of SC-PCR approaches, molecu-lar phylogenetic analyses of SSU rDNA sequences, andcomparative morphological data demonstrated the cellular

Mar Biodiv (2013) 43:133–147 145

identity of a previously unidentified environmental SSUrDNA sequence clade and enabled us to establish four newspecies within one novel genus: Polyplicarium lacrimae n.gen. et sp. (type species), P. curvarae n. gen. et sp., P.translucidae n. gen. et sp., and P. citrusae n. gen. et sp.These data highlight significant limitations of environmentalPCR surveys of biodiversity, mainly that accurate interpre-tations of the resulting DNA sequences require a compre-hensive sample of reference species that have also beencharacterized at the cellular level. Hopefully, an appreciationfor this organismal context will inspire future explorationinto the overall diversity of marine gregarines using anapproach that combines single-cell PCR, molecular phylo-genetic analyses, and comparative morphology.

Acknowledgments This research was supported by grants from theNational Science and Engineering Research Council of Canada(NSERC 283091-09) and the Canadian Institute for AdvancedResearch, Program in Integrated Microbial Biodiversity.

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